Enzymes are used in various types of industrial processes in the pulp and paper industry, textile industry, and in the food, feed and beverage industry. Enzymes are also used in the cosmetic and pharmaceutical industry and in detergents. The industrial enzymes can be of animal, plant or microbial origin, and extracellular enzymes are preferred. They are usually more stable and can easily be produced by recombinant technology. The isolation and purification of intracellular enzymes from the host cells is costly and laborious, and therefore it is an advantage, if the enzyme is extracellular i.e. a protein that is secreted from the cell. Further it is desirable that the enzyme can be produced in great amounts in an industrial scale and that the organism is safe, and easy and economic to cultivate.
Proteins are significant constituents in renewable raw materials and thus food, textile fibres etc contain significant amounts of protein. Enzymes can be used for modification of the proteins and their technological properties in these materials. Protein matrix can be modified by hydrolytic enzymes (proteases) being able to decrease the molecular weight of the protein. Proteins can also be modified by enzymes being able to create covalent cross-links between amino acid residues in proteins (as an example transglutaminase, which creates isopeptide links between lysine and glutamine residues) or by enzymes being able to oxidize certain amino acid residues. Oxidation of certain amino acid residues can in turn also result in formation of cross-links. Modification of proteinaceous material by cross-linking is frequently used e.g. in food processing. Regarding food quality, texture is a very essential factor. It is not only related to sensory perception but also to water holding capacity, gelling and emulsifying properties and stability.
Enzyme-aided structure engineering via protein cross-linking can be exploited in several food applications, e.g. in meat, fish, dairy and cereal processing. Transglutaminase is a well known enzyme e.g. for cold-binding of meat parts together to produce restructured meat products, for texture improvement and water holding capacity of minced meat products, improvement of structure of fish raw materials, milk gel forming in yoghurt production with better water holding without undesirable syneresis effect, prevention of texture deterioration of pasta products after cooking, improved loaf volume of bread baked from low-grade flours. Enzymatic cross-linking of vegetarian foodstuff with e.g. transglutaminase is disclosed in WO 03/007728.
Transglutaminase is known to catalyse cross-linking in or between proteins via formation of ε(γ-glutamyl) lysine isopeptide bonds in/between different proteins such as myosin, gelatine and collagen, casein, caseinate, whey protein, soy protein, gluten, egg proteins (Kuraishi et al., 2001; Nielsen, 1995). The reactivity is, however, dependent on the availability and accessibility of the target amino acids, i.e. lysine and glutamine in the protein substrate. Thus, not all proteins are suitable substrates for transglutaminase due to insufficient accessibility or limited quantity of glutamine or lysine residues in the protein.
Phenol oxidases using oxygen as an electron acceptor are particularly suitable for enzymatic processes as no separate cofactors needing expensive regeneration i.e. NAD(P)H/NAD(P) are required in the reactions. These phenol oxidases include e.g. laccase and tyrosinase. They are both copper proteins and can oxidize various phenolic compounds. The substrate specificity of laccases and tyrosinases is partially overlapping.
Tyrosinase catalyses both the o-hydroxylation of monophenols and aromatic amines and the oxidation of o-diphenols to o-quinones or o-aminophenols to o-quinoneimines (Lerch, 1981). Traditionally tyrosinases can be distinguished from laccases on the basis of substrate specificity and sensitivity to inhibitors. However, the differentiation is nowadays based on structural features. Structurally the major difference between tyrosinases and laccases is that tyrosinase has a binuclear copper site with two type III coppers in its active site, meanwhile laccase has altogether four copper atoms (type I and II coppers, and a pair of type III coppers) in the active site.
Tyrosinase is capable of oxidising tyrosine residues in proteins to the corresponding quinones, which can further react with e.g. free sulfhydryl and/or amino groups resulting in formation of tyrosine-cysteine and tyrosine-lysine cross-links (Ito et al., 1984). Quinones have also been suggested to form tyrosine-tyrosine linkages by coupling together.
Methods for cross-linking proteins by laccases have been disclosed e.g. in US2002/9770. Plant proteins derived from beans and cereals and animal proteins including milk, egg, meat, blood and tendon are listed as suitable substrates. However, laccases form radicals to proteins and also to other possible substrates (e.g. phenolic components). Therefore the process is more difficult to control than quinone-derived non-radical reactions catalyzed by tyrosinase. In the laccase-catalyzed reaction also some stable radicals can retain in the matrix causing depolymerization and subsequent disruption of the matrix as a function of time. Fungal laccases are disclosed in US2002/19038.
The ability of tyrosinase to cross-link food proteins has been reviewed (Matheis and Whitaker, 1984; Matheis and Whitaker, 1987). In these studies intracellular Agaricus tyrosinase has been used. The cross-linking of proteins with tyrosinase proceeds via the formation of o-quinones from protein-bound tyrosine. These o-quinones either condense with each other or react with free amino and sulfhydryl groups present in proteins.
Tyrosinases have been suggested for use in cross-linking of whey proteins (Thalmann and Loetzbeyr, 2002) and in modifying the physical properties of dough (Takasaki and Kawakishi, 1997). In addition to food protein applications tyrosinases may be used e.g. in the cosmetic and pharmaceutical field (DE 102 44 124). WO99/57993 discloses the use of cross-linking enzymes in ruminant feed, and US2003/0177589 discloses a method of treating proteinaceous fibres with a tyrosinase enzyme, thereby preventing e.g. shrinkage of wool textiles. Conjugates obtained by contacting a polypeptide such as gelatine and a polysaccharide such as chitosan with a tyrosinase is disclosed in WO2004/029096. The gelatine-chitosan conjugate can be used in medical applications. Tyrosinase has also been used to polymerise tropocollagen macromolecules, which are the constituents of collagen fibres (Dabbous, 1966). Formation of inter- and intramolecular cross-links between tyrosine residues resulted in polymerisation.
Tyrosinases are widely distributed in nature. They are related to melanin and eumelanin synthesis in plants, mammals, and insects. In fruits and vegetables tyrosinase is responsible for enzymatic browning reactions and in mammals for pigmentation. In fungi the role of tyrosinase is correlated with cell differentiation, spore formation, virulence and pathogenesis (Sanchez-Ferrer et al., 1995).
The best known and characterized tyrosinases are of mammal origin. The most extensively investigated fungal tyrosinases both from a structural and functional point of view are from Agaricus bisporus (Wichers et al., 1996) and Neurospora crassa (Lerch, 1983). Also a few bacterial tyrosinases have been reported, of which Streptomyces tyrosinases are the most thoroughly characterized (U.S. Pat. No. 5,801,047 and U.S. Pat. No. 5,814,495). In addition, tyrosinases have been disclosed e.g. from Bacillus and Myrothecium (EP 919 628), Mucor (JP 61115488), Miriococcum (JP 60062980) Aspergillus, Chaetotomastia, Ascovaginospora (Abdel-Raheem and Shearer, 2002), Trametes (Tomsovsky and Homolka, 2004).
Intracellular fungal tyrosinases have been described and they are supposed to be cytoplasmic enzymes (Van Gelder et al., 1997). Indeed the fungal tyrosinase genes analyzed so far do not have a signal sequence, although there are reports claiming that tyrosinase activity has been detected in culture supernatant of some freshwater ascomycetes (Abdel-Raheem and Shearer, 2002), Chaetomium (JP 62205783) and Trametes spp. (Tomsovsky and Homolka, 2004). The reported tyrosinase activities in culture supernatants can be due to cell autolysis.
Phenol oxidase and peroxidase production during interspecific interactions between two Basidomycetes (Serpula lacrymans and Conidiophora puteana and several Deuteromycetes (Trichoderma spp. and Scytalium FY) have been investigated by Score et al., 1997 by preliminary and simple plate analysis. The authors used naphtol and p-cresol as specific substrates for laccases and tyrosinases, respectively. Laccase was detected in the interactions involved in Serpula lacrymans and all three Trichoderma isolates. Indeed, Hölker et al. 2002 have recently isolated and characterized laccase from Trichoderma. Based on results from the preliminary plate tests also tyrosinase activity was suggested in the tested Trichoderma species (Score, 1997). However, no tyrosinase was isolated or purified. Mackie et al., 1999 have reported laccase and tyrosinase activity in Trichoderma viride, when studying volatile organic compound interactions between soil bacterial and fungal isolates. However, so far tyrosinases have not been isolated nor further characterized from Trichoderma. 
Streptomyces is reported to have an extracellular tyrosinase and secretes the enzyme to culture supernatant, however, the tyrosinase enzyme itself does not have a signal sequence for secretion. The secretion of Streptomyces tyrosinase requires a second protein (called MelC1 in S. antibioticus) that has a signal sequence (Leu et al., 1992; Tsai and Lee, 1998), and this makes the industrial production of Streptomyces tyrosinase more tedious and complicated than production of a naturally secreted tyrosinase.
Microbial tyrosinases have been produced heterologously. Two tyrosinase genes from e.g. Agaricus bisporus have been expressed in small amounts in E. coli (Wichers et al., 2003). A tyrosinase gene melO from Aspergillus oryzae has been produced heterologously in Saccharomyces cerevisiae (Fujita et al., 1995). In addition a tyrosinase gene from Streptomyces antibioticus was coexpressed in E. coli with an ORF438 protein probably involved in protein secretion (Della-Cioppa et al., 1990, U.S. Pat. No. 5,801,047).) However, the expression levels of microbial tyrosinases reported in literature are relatively low, and will not allow high titre production of the enzyme. Indeed the availability of tyrosinase has restricted testing of the enzyme in different applications. In practice the Agaricus tyrosinase available from Sigma has been the only commercially available tyrosinase. This commercial enzyme is, however, a crude enzyme with relatively low activity and it is very expensive.
In view of the above, there is still a need for novel tyrosinases that have desirable properties both with respect to activity and availability. For easy recovery, the enzyme should be secreted out of the cell in high amounts, whereby the need for cell disruption is avoided in the isolation process and complications arising from cellular debris can be avoided. The enzyme should further be suitable for production by recombinant technology in commercially acceptable quantities, economically and with minimum environmental and health risks. The use of safe organisms is especially important in food applications. The present invention responds to these demands.
Although intracellular proteins could in principle also be produced in recombinant systems as secreted products by coupling them to a signal sequence, naturally secreted proteins are expected to be much more favourable for extracellular production. This is because they are well adapted to the protein folding and trafficking machineries of the secretory pathway.